A botulinum toxin produced by Clostridium botulinum, anaerobic Gram-positive bacteria, is the most lethal neurotoxin on earth. It is classified into seven types, A, B, C, D, E, F and G, and the property of each type has been elucidated. The types are distinguishable from each other by respective type-specific neutralizing antibodies. Depending on the types, a botulinum toxin may vary in animal species it may affect, severity of paralysis it induces, duration of time of its action, and the like. An active center protein of a botulinum toxin has a molecular weight of about 150 kDa (NTX) as common in all the known seven types.
Any botulinum toxin, when produced from Clostridium botulinum, is in a molecular form of a complex composed of NTX and relevant nontoxic proteins. A type A botulinum toxin is produced in a molecular form of either 900 kDa (LL toxin), 500 kDa (L toxin), or 300 kDa (M toxin) (FIG. 1). These LL, L and M toxins are called a botulinum toxin complex or a progenitor toxin. The botulinum toxins are separate to release NTX and NTNH (a nontoxic non-HA protein) under alkaline conditions (pH 7.2 or higher). By utilizing this property, it is possible to isolate NTX of 150 kDa (an active center protein that endows a neurotoxin with the activity; also called “S toxin”) alone.
The botulinum toxins are, upon absorption in the upper small intestine, separate to release nontoxic proteins and a neurotoxin in a lymphatic vessel. The released neurotoxin is then bound to a receptor at the nerve end at its C-terminus of a heavy chain and taken into neurons via the receptor. Then, it specifically cleaves a protein in the presynaptic membrane through a light chain zinc methaloendopeptidase activity and inhibits a calcium-dependant release of acetylcholine to thereby block neuromuscular transmission at the synapse (Non-patent reference 1).
Although a botulinum toxin is a neurotoxin that may lead human to death in botulinum intoxication through blockage of systemic neuromuscular transmission, it may also be utilized as a remedy for treating a disease with a muscle overactivity such as e.g. dystonia by positively making use of its activity and by administering directly into the muscle of a patient suffering from the disease so that a local muscular tension may be relieved (Non-patent reference 2). For instance, a type A botulinum toxin complex (Allergan Inc., BOTOX®; has been approved as a medicament for treating blepharospasm, strabismus, hemifacial spasm, and cervical dystonia, and for treating wrinkles at the middle of the forehead by the Food and Drug Administration (FDA). A type B botulinum toxin complex (Elan Pharmaceuticals, MYOBLOC®; has also been approved as a medicament for treating cervical dystonia by FDA. It is said that a type A botulinum toxin has a higher potency and a longer duration of action as compared to types other than a type A botulinum toxin. An average duration of action of a type A botulinum toxin from its single intramuscular administration up till amelioration of symptoms is typically about 3 to 4 months.
Therapeutic preparations of botulinum toxin are available from Allergan Inc. (U.S.A.), Ipsen Limited (U.K.) or Elan Pharmaceuticals (Ireland). These commercially available therapeutic preparations of botulinum toxin consist of a purified botulinum toxin complex (LL toxin) alone in a molecular structure with bound relevant nontoxic proteins. For instance, the currently commercially available therapeutic preparations of type A botulinum toxin, i.e. BOTOX® from Allergan Inc. and Dysport® from Ipsen Limited, consist of an LL toxin of a botulinum toxin complex comprising as its component an HA protein such as HA17, HA34, or HA70.
In recent years, type A NTX preparations (Merz Pharma, Xeomin®, Germany) comprising no nontoxic proteins were sold in 2005, similar other preparations underwent clinical trials in the U.S.A. and development of next-generation preparations has actively been done.
On the other hand, a botulinum toxin isolated from patients suffering from infant botulism in 1990, though belonging to type A, consists of M toxin with no HA proteins (HA-negative). Type A Clostridium botulinum that produces M toxin with no HA protein has been first identified in Japan in 1986 from patients suffering from infant botulism (Non-patent reference 3). The clinically isolated strains include Kyoto-F, Chiba-H, Y-8036, 7I03-H, 7I05-H and KZl828. When compared with the other types A to G of botulinum toxins, a botulinum toxin from Clostridium botulinum that causes infant botulism is a peculiar neurotoxin distinct from any types of these toxin molecules.
From the genetic point of view, a genetic mechanism of Clostridium botulinum as infant botulism pathogen is different from those of the other types of botulinum toxin. Most of the conventional botulinum toxins, typically type A botulinum toxin, has been seen as a botulinum toxin complex having Haemagglutinin (HA) protein as a component thereof. Genes coding for HA proteins such as HA17, HA34 and HA70 are contained in neurotoxin genes of types A, B, C, D and G Clostridium botulinum but are completely absent in those of Clostridium botulinum as infant botulism pathogen. Also, genes of Clostridium botulinum as infant botulism pathogen contain a regulator gene such as p47 (Non-patent reference 4). Besides, it was shown that a sequence of the NTNH protein of botulinum toxin produced by Clostridium botulinum as infant botulism pathogen is a miscellany, i.e. a mosaic, of nontoxic non-HA protein NTNH genes of type C and type A (Non-patent reference 5 and Non-patent reference 6).
As for NTX molecules per se, a molecular weight is distinct from each other in that a heavy chain of the conventional type A botulinum toxin is 93 kDa whereas botulinum toxin produced by Clostridium botulinum as infant botulism pathogen is 101 kDa. They also show different protease reactivity (Non-patent reference 7). The amino acid sequences of these two isotypes of the botulinum toxins are different by 10.1% as a whole and, by 13% in the heavy chain region and by as low as 4.9% in the light chain region (Non-patent reference 8).
It is reported that cell strains used for manufacturing commercially available preparations of type A botulinum toxin are HALL strain for BOTOX® and Xeomin® and NCTC2916 strain for Dysport® (Non-patent reference 9 and Non-patent reference 10), which may be classified into type A botulinum toxin which comprises HA protein, i.e. type A1 botulinum toxin. On the other hand, botulinum toxin produced by Clostridium botulinum as infant botulism pathogen may be classified into type A2 botulinum toxin.
In recent years, a problem has been presented that repetitive administration of botulinum toxin may induce production of an anti-botulinum toxin antibody to diminish efficacy of the botulinum toxin. For instance, it is reported that an antibody induction in BOTOX® is 3 to 10% (Non-patent reference 11). It is pointed out that, as one of the causes, HA contained in therapeutic preparations has an adjuvant activity for antibody production (Non-patent reference 12), which adjuvant activity is thought to facilitate production of a neutralizing antibody to NTX.
For a highly purified botulinum toxin, it was formerly reported by Tse C K., et al. (Non-patent reference 13) and also in WO1996/11699 (Patent reference 1) as to a process for purification (p. 6, line 9 to p. 7, line 2) and pharmaceutical compositions (p. 11, Table 2).    Patent reference 1: WO1996/11699    Non-patent reference 1: Jankovic J. et al., Curr. Opin. Neurol., (7): p. 358-366, 1994    Non-patent reference 2: Ryuji Kaji et al., “Dystonia and botulinum therapy”, Shindan-To-Chiryosha, 2005    Non-patent reference 3: Sakaguchi G. et al., Int. J. Food Microbiol., 11: p. 231-242, 1990    Non-patent reference 4: Kubota T. et al., FEMS Microbiology letters., 158: p. 215-221, 1998    Non-patent reference 5: Kubota T. et al., Biochem. Biophys. Res. Commun., 224(3): p. 843-848, 1996    Non-patent reference 6: Sakaguchi G. et al., Int. J. Food Microbiol. 11: p. 231-242, 1990    Non-patent reference 7: Kozaki S. et al., Microbiol. Immunol. 39(10): p. 767-774, 1995    Non-patent reference 8: Cordoba J. et al., System. Appl. Microbiol. 18: p. 13-22, 1995    Non-patent reference 9: Ryuji Kaji et al., “Dystonia and botulinum therapy”, Shindan-To-Chiryosha: 23, 1996    Non-patent reference 10: Dressler D. et al., Disabil Rehabil. 29(23): p. 1761-1768, 2007    Non-patent reference 11: Brin M F., Muscle Nerve Suppl., 6: p. 146-168, 1997    Non-patent reference 12: Arimitsu H. et al., Infect. Immun., 71(3): p. 1599-1603, 2003    Non-patent reference 13: Tse C K. et al., Eur. J. Biochem., 122(3): p. 493-500, 1982